Cloning, Expression, Purification And Identification Of Im58, Potassium Channel Blocker From The Scorpion Isometrus Maculates

Scorpion is one of the oldest species which can produce peptides and proteins as theactive material of scorpion venom in the long evolutionary process. It survived more than fourhundred million years.What is more,the scorpion venom that contains many toxic peptideswith20～80amino acid residues would play an important role for the scorpion’s capture anddefence. According to the report, there are more than100000kinds of scorpion venompeptides. Most of these scorpion toxins can interact with ion channels that are nearly350kinds of membrane channel subtypes. Specially, sodium, potassium, calcium, chloride ionchannels are well known. As we know,ion channels can keep on metabolic activity andexchange environment of matter or energy within cells.However, the abnormal coding geneexpression of ion channels will produce ion channel diseases which are characterized byweakening or strengthening the function of ion channels. In recent years,the ion channel hasbecome the new targets for drug development. Among all kinds of ion channels, potassiumchannels are a diverse and the most complicated family of membrane proteins in present.potassium channels widely expressed in the plants, animals, and even unicellular prokaryotes,therefore, they play important roles in physiological activities. Most of channelopathies thathave been associated with nerve, heart and muscles are resulted from abnormal expression orstructure of potassium channels. Present research focuse on potassium Kv1.3channels whichare related to autoimmune diseases. Previous research showed that the autoimmune diseasewould lead to T cell’s activation, proliferation and differentiation.Besides, over expressionKv1.3potassium channels in T cells were linked to autoimmune diseases. Therefore, Kv1.3channels have become a novel target for the treatment of autoimmune diseases. Looking forspecific Kv1.3potassium channel inhibitors has become the current hot spot for exploitationmedicines to treat autoimmune disease.In this thesis, we selected a scorpion toxin Im58gene from the venomous gland cDNAlibrary of Chinese Isometrus maculatus from Hainan province. The cDNA sequence of Im58contains a180bp open reading frame (ORF) which encods60amino acids, including thesignal peptide of22residues and the mature peptide of38residues. Besides, the secondarystructure of Im58consist of an α helix and three β sheets being linked through three disulfidebridges.The full cDNA sequence of Im58was obtained by means of overlap PCRamplification. Through the following steps, we successfully constructed the recombinantvector of pGEX-4T-1-Im58by digestion, connection and transformation, then transformedconstructed vector to E.coli/Rosetta to express the fusion protein GST-Im58which be induced with IPTG.. Fusion protein GST-Im58was purified by GSH affinity chromatography thendigested by enterokinase and purified by RP-HPLC.Finally,we calculated the molecularweight of mature Im58peptide through MALDI-TOF–MS.Whole cell patch-clamp experiments were used to identify the effect of Im58on Kv1.2channels and Kv1.3channels.Electrophysiological experiments results indicated that Im58could block about30%of Kv1.2channel currents even at the concentration of100nM.However, Im58was found to potentially block the Kv1.3channels with the IC50of2.3±0.83nM.Therefore, as a new Kv1.3channel blockers, Im58peptide will be used as theprecursor drug for theraping autoimmune diseases.To detect the interaction mechanism between Im58and Kv1.3channels, we simulatedthe3D-structure of Im58by means of SWISS-MODEL software, and compared the proteinsequence of Im58to the other α-KTx family toxins through online Blast. Computation resultsshowed that Im58had classic’function dyad’ which was made up of a lysine and its distanceof about6aromatic amino acids.Therefore, we speculate that the lysine in’function dyad’may the pivotal amino acid residues of IM58to block the channel pore of Kv1.3. To confirmthe speculation, in the nest step, we plan to design and renovate Im58to resolve theinteracting mechanism of Im58and Kv1.3channels.